In a previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 70, 2583–2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Galβ1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new β1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such β1-3galactosyltransferase activity, which can transfer galactosyl residues with the β1-3 linkage to β1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc2GlcNAc2Man3GlcNAc2-PA as acceptor N-glycan, we detected the β1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique β1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.